UNVEILING MOLECULAR PATHWAYS DISRUPTED IN SMS AND SMS-LIKE PATIENTS BY MEANS OF GENOMIC AND FUNCTIONAL APPROACHES

Smith Magenis Syndrome (SMS, OMIM#182290) is a complex genomic disorder with incidence of 1:15000-25000, clinically characterized by neurological abnormalities with variable intellectual disability (ID), craniofacial dysmorphisms, behavioral and sleep disturbances, and speech and motor delay. SMS is caused by haploinsufficiency of RAI1 (Retinoic Acid-Induced 1) gene to either large 17p11.2 deletion (90%) or point mutations/intragenic microdeletions (10%). RAI1 encodes a transcription factor, working as chromatin reader in a regulatory complex, that positively regulates the expression of many neurodevelopmental and circadian rhythm genes. However, only 50% of individuals with SMS clinical suspicion is confirmed by the genetic test, suggesting that other loci may be involved directly or indirectly in the same pathway of RAI1 gene, hence contributing to the SMS-like phenotype. In order to deepen the genetic mechanisms underlying the SMS spectrum, we analyzed a cohort of 30 patients with SMS clinical diagnosis through several genetic approaches. High resolution array-CGH, performed to pinpoint rare pathogenic CNVs containing dosage sensitive genes that might be implicated in RAI1 molecular pathways, disclosed 4 pathogenic CNVs. Further studies, namely RAI1 NGS sequencing, MLPA, and RT-qPCR revealed in one patient multiple peculiar molecular RAI1 defects consistent with SMS molecular diagnosis. Considering that sleep disturbance is a main feature of SMS and the circadian genes oscillatory expression can be observed in peripheral melatonin target tissues, an additional aim of this project was the evaluation of circadian rhythm genes expression in peripheral blood cells of selected 16 SMS/SMS-like patients through RT-qPCR analysis. Among the tested genes, CLOCK, BMAL2, PER2, and NR1D1 were found dysregulated in at least one patient analyzed. Thus, this study turned out to be an useful preliminary approach to evaluate any circadian dysfunctions in patients with sleep disturbance. Another crucial aim of this thesis was the investigation of candidate genes implicated in RAI1 molecular pathway through a functional approach. Considering that neurodevelopmental disorders (NDDs) are extraordinarily difficult to study, we used human iPSCs differentiated into neurons, in the attempt to elucidate the molecular alterations that give rise to SMS. A male patient of our cohort, bearing a rare 54 kb maternal deletion at Xq13.3 mapping 29 kb far from 5’UTR of ZDHHC15 gene that results downregulated in blood, was selected for iPSCs reprogramming and differentiation in cortical neurons. Starting from peripheral blood mononuclear cells we successfully obtained three and two genomic stable iPSCs clones of patient and his parents, respectively. Patient’s iPSCs were comparable to parents’ iPSCs for morphology, pluripotency-related markers expression, and capability of embryo body formation and spontaneous differentiation into the three germ layers. The subsequent iPSCs differentiation produced cortical neurons found positive to several specific neuronal markers. Although, we were unable to determine the amount of ZDHHC15 mRNA on mature cortical neurons, an altered functional activity of patient’s cultured iPSCs-derived neurons was disclosed. The evaluation of electrophysiological profiles of differentiated neurons revealed in the patient and his mother compared to the father the absence of a progressive increase in mEPSCs frequency and amplitude, resembling a defected excitatory synaptic development. To sum up, the present combined approach using both genomic and functional techniques resulted an efficient strategy to deepen the molecular and genetic mechanisms underlying SMS-like phenotype, and to increase the possibility to uncover the networks of genes underlying NDDs.